Several factors influence the engraftment of allogeneic hematopoietic stem cells including host immune status, histocompatibility, hematopoietic space, stem cell dose, and subsets and doses of T cells within the allograft. Host immune status has not been emphasized, relative to allogeneic stem cell engraftment, when myeloablative conditioning regimens are utilized for allogeneic HSCT, as they markedly attenuate the HVG response that mediates graft rejection. However, when non-myeloablative and reduced-intensity conditioning regimens are utilized prior to allogeneic HSCT, it has been both observed in murine models and inferred from clinical trials data that engraftment is inversely correlated with host immune status. As there can be significant variability in the immune status of patients being considered for alloHSCT, we surmised that individual patients required different levels of host immune depletion in order to achieve the rapid and complete engraftment of allogeneic hematopoietic stem cells, especially if they were from someone other than an HLA-matched sibling. To address the problem of host immune status variability, we developed a novel treatment approach, referred to as targeted immune-depletion (TID), which utilizes an induction regimen consisting of repetitive cycles of disease-specific immunosuppressive chemotherapy at conventional doses that is administered until a pre-determined level of host immune depletion, as determined by circulating T cell numbers, has been achieved. The induction regimen is followed by a reduced-intensity conditioning regimen consisting of cyclophosphamide and fludarabine. This regimen has been held constant throughout all of our clinical trials to permit comparisons to other studies. Targeted immune-depletion: 1) permits host T cell depletion in a patient-specific manner, 2) provides disease control, and 3) utilizes chemotherapy at conventional doses thereby minimizing toxicity. The targeted immune depletion approach has been implemented through a systematic, step-wise progression of studies in clinical settings in which one of the barriers to engraftment (e.g. T cell depletion, HLA-disparity) was increased, while other engraftment variables were kept constant. In the first clinical setting, patients received T cell replete allografts from HLA-matched siblings. In the second setting, patients received allografts from which the T cells were removed, or depleted, from their HLA-matched siblings, and in the third clinical setting, patients received T cell depleted allografts from HLA-mismatched related donors. In our first trials, we investigated targeted immune-depletion in the first clinical setting in which patients received a conventional, T cell replete (TCR) allograft from an HLA-matched sibling. The results from the first trial, 99-C-0143, demonstrated that sustained, complete donor engraftment in both myeloid (CD33+) and T lymphoid (CD3+) compartments could be achieved rapidly after targeted immune depletion, as determined by chimerism studies for donor and host elements by 14 days after transplantation. These results were similar to those achieved with a conventional, myeloablative conditioning regimen. The subsequent trial with TCR allografts from HLA-matched siblings, 03-C-0077, tested targeted immune depletion with the addition of methotrexate immediately post-transplant to reduce GVHD; methotrexate eliminates activated T cells from the stem cell graft in vivo, which can affect stem cell engraftment. The results of this trial again demonstrated 100% donor engraftment with complete donor chimerism in the overwhelming majority (>90%) of patients by day +28 post-transplant. Based on the results in the first clinical setting, we then proceeded to test the targeted immune depletion approach in the second clinical setting where the allograft was T cell depleted to further reduce the risk of graft-versus-host disease, but increasing the risk of graft rejection. In this trial, 00-C-0119, results were similar to those observed in the first clinical setting. The targeted immune depletion approach led to the rapid establishment of full donor chimerism in the majority of patients. There were no graft rejections. There was, however, evidence of mixed lymphoid chimerism in a minority of patients. The early presence of mixed lymphoid chimerism correlated statistically with the patients pre-transplant circulating T cell levels. It did not correlate with the amount of prior therapy that patients had received prior to transplant, which had been correlated with engraftment in other reports of reduced-intensity conditioning. This study demonstrated that the degree of donor chimerism correlated inversely with pre-transplant circulating T cell numbers, confirming the concept and value of targeted immune depletion. We have demonstrated that the targeted immune depletion of host T cell permits the rapid and complete engraftment of allogeneic stem cells, abrogating a barrier of graft rejection. We also demonstrated that circulatory T cell numbers are a surrogate determinant of graft rejections risk and that targeted immune depletion may permit an individualized approach to conditioning based on host immune status prior to allogeneic HSCT. As part of this rational, incremental approach, we are currently testing targeted immune depletion in the following areas: A. HLA-matched sibling donors - Based on the above results, our current trials that utilize stem cells from HLA-matched siblings are investigating whether we can further reduce the intensity of the conditioning regimen, relying more on the immune depleting effects of the induction regimen, thus further reducing therapy-related toxicities to a level associated with conventional chemotherapy. Potentially, the entire transplantation process could primarily be performed in an outpatient setting and a significant proportion of the process could be administered in a community setting. Preliminary clinical results indicate that this is possible, representing a paradigm shift in the approach to the engraftment of allogeneic stem cells. B. HLA-matched unrelated donors - The use of stem cells from HLA-matched unrelated donors increases the availability of allogeneic cellular therapy to an additional 30-50% of patients. Transplantation from HLA-matched unrelated donors is associated with higher degrees of graft rejection and mixed chimerism due to differences in minor histocompatibility antigens between donor and recipient. As part of our sequential approach, I am about to initiate a clinical trial utilizing T cell replete allografts from HLA-matched unrelated volunteer donors utilizing the targeted immune depletion approach to study its effect upon engraftment. This is an inter-institutional (NCI, NHLBI, and NIAID), interagency (NIH and DOD), and extramural (National Marrow Donor Program) collaboration. It is projected that our initial trial will be completed within two to three years. C. Haplo-identical related donors - Haplo-identical donors are available to over 90% of patients; however, due to HLA-disparity, transplantation with stem cells from these donors is associated with high degrees of graft rejection, GVHD, and post-transplant immunodeficiency. T cell depletion of the stem cell graft is considered essential in haplo-identical HSCT to prevent GVHD; however, it further in [summary truncated at 7800 characters]